Leiden University Medical Center, Department of Molecular Cell Biology, Leiden, The Netherlands.
J Gene Med. 2009 Nov;11(11):990-1004. doi: 10.1002/jgm.1395.
Many studies aimed at retargeting adenovirus (Ad) rationally focus on genetic modification of fiber, which is the primary receptor-binding protein of Ad. Retargeted fibers ultimately require functional validation in the viral context.
Lentiviral vectors (LV) were used to express fiber variants in cells. Infections with a fiber gene-deleted Ad vector yielded fiber-pseudotyped viruses. An enzyme-linked immunosorbent assay and slot blot-based assays probed target binding-ability of retargeted fibers. Differential treatments with an alkylating agent prior to western blot analysis allowed for examination of intra- and extracellular redox states of fibers.
In the present study, LV-based fiber-pseudotyping of Ad is presented as an accelerated means to test new fibers. LV-mediated gene transfer yielded stable and uniform populations of fiber variant-expressing cells. These populations were found to effectively support fiber-pseudotyping of Ad. As a secondary objective of the study, we functionally assessed a chimeric fiber harboring a tumor antigen-directed single-chain antibody fragment (scFv). This fiber was shown to trimerize and achieve a degree of binding to its antigenic target. However, its capsid incorporation ability was impaired and, moreover, it was unable to confer a detectable level of target binding upon Ad. Importantly, subsequent analyses of this fiber revealed the improper folding of its scFv constituent.
LV-based fiber-pseudotyping was established as a convenient method for testing modified fibers for functionality within Ad particles. Furthermore, a new chimeric fiber was found to be inadequate for Ad retargeting. The folding difficulties encountered for this particular fiber might be generally inherent to the use (i.e. for genetic Ad capsid incorporation) of complex, disulfide bridge-containing natural ligands.
许多旨在合理靶向腺病毒 (Ad) 的研究都集中在纤维的遗传修饰上，纤维是 Ad 的主要受体结合蛋白。靶向纤维最终需要在病毒环境中进行功能验证。
慢病毒载体 (LV) 用于在细胞中表达纤维变体。用缺失纤维基因的 Ad 载体感染产生纤维假型病毒。酶联免疫吸附试验和斑点印迹试验检测靶向纤维的靶结合能力。在用烷基化剂进行 Western blot 分析之前进行差异处理，以检查纤维的细胞内和细胞外氧化还原状态。
在本研究中，我们提出了基于 LV 的 Ad 纤维假型作为测试新纤维的加速手段。LV 介导的基因转移产生了稳定且均匀的纤维变体表达细胞群体。这些群体被发现能够有效地支持 Ad 的纤维假型。作为研究的次要目标，我们功能评估了一种携带肿瘤抗原导向的单链抗体片段 (scFv) 的嵌合纤维。该纤维被证明能够三聚化并达到与其抗原靶标的一定结合程度。然而，其衣壳掺入能力受损，此外，它无法赋予 Ad 可检测水平的靶结合。重要的是，对该纤维的后续分析揭示了其 scFv 组成部分的不正确折叠。
LV 基于纤维假型被确立为一种方便的方法，用于测试 Ad 颗粒中功能性修饰的纤维。此外，发现一种新的嵌合纤维不适合 Ad 靶向。该纤维遇到的折叠困难可能普遍存在于复杂的、含有二硫键的天然配体的使用（即用于遗传 Ad 衣壳掺入）中。
